Nesfatin-1 is a recently identified satiety molecule detectable in neurons of the hypothalamus and nucleus of solitary tract (NTS). Immunohistochemical studies revealed nesfatin-1-immunoreactive (irNEF) cells in the Edinger-Westphal nucleus, dorsal motor nucleus of vagus, and caudal raphe nuclei of the rats, in addition to the hypothalamus and NTS reported in the initial study. Double-labeling immunohistochemistry showed that irNEF cells were vasopressin or oxytocin positive in the paraventricular and supraoptic nucleus; cocaine-amphetamine-regulated transcript or tyrosine hydroxylase positive in arcuate nucleus; cocaine-amphetamine-regulated transcript or melanin concentrating hormone positive in the lateral hypothalamus. In the brainstem, irNEF neurons were choline acetyltransferase positive in the Edinger-Westphal nucleus and dorsal motor nucleus of vagus; tyrosine hydroxylase positive in the NTS; and 5-hydroxytryptamine positive in the caudal raphe nucleus. The biological activity of rat nesfatin-1 (1-82) (100 nm) was assessed by the Ca(2+) microfluorometric method. Nesfatin-1 elevated intracellular Ca(2+) concentrations [Ca(2+)](i) in dissociated and cultured hypothalamic neurons. The response was prevented by pretreating the cells with pertussis toxin (100 nm) or Ca(2+)-free solution and by a combination of the L-type and P/Q-type calcium channel blocker verapamil (1 microm) and omega-conotoxin MVIIC (100 nm). The protein kinase A inhibitor KT 5720 (1 microm) suppressed nesfatin-1-induced rise in [Ca(2+)](i). The result shows that irNEF is distributed to several discrete nuclei in the brainstem, in addition to the hypothalamus and NTS reported earlier, and that the peptide interacts with a G protein-coupled receptor, leading to an increase of [Ca(2+)](i), which is linked to protein kinase A activation in cultured rat hypothalamic neurons.
Salvinorin A, acrylamido]morphinan hydrochloride), and 3FLB (diethyl 2,4-di-[3-fluorophenyl]-3,7-dimethyl-3,7-diazabicyclo[3.3.1]nonane-9-one-1,5-dicarboxylate) are structurally distinctly different from U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide methanesulfonate], the prototypic selective agonist. Here, we investigated their in vitro pharmacological activities on receptors expressed in Chinese hamster ovary cells and in vivo antiscratch and antinociceptive activities in mice. All three compounds showed high selectivity for the opioid receptor (KOR) over the opioid receptor (MOR) and ␦ opioid receptor (DOR) and nociceptin or orphanin FQ receptors. In the guanosine 5Ј-O-(3-[35 S]thio)triphosphate ([ 35 S]GTP␥S) binding assay, all three were full agonists on the KOR. The rank order of affinity and potency for the KOR was TRK-820 Ͼ Ͼ U50,488H ϳ salvinorin A Ͼ Ͼ 3FLB. TRK-820 acted as a partial agonist on MOR and DOR, whereas salvinorin A and 3FLB showed no activities on these receptors. Salvinorin A, TRK-820, and 3FLB caused internalization of the human KOR in a dosedependent manner. Interestingly, although salvinorin A and U50,488H had similar potencies in stimulating [ 35 S]GTP␥S binding, salvinorin A was about 40-fold less potent than U50,488H in promoting internalization. Following 4-h incubation, all three compounds induced down-regulation of the human KOR, with salvinorin A causing a lower extent of down-regulation. Although TRK-820 was potent and efficacious against compound 48/80-induced scratching, salvinorin A showed low and inconsistent effects, and 3FLB was inactive. In addition, salvinorin A and 3FLB were not active in the acetic acid abdominal constriction test. The discrepancy between in vitro and in vivo results may be due to in vivo metabolism of salvinorin A and 3FLB and possibly to their effects on other pharmacological targets.At least three types of opioid receptors, , ␦, and , mediate pharmacological effects of opioid drugs and physiological actions of endogenous peptides (for review, see Chang, 1984;Mansour et al., 1988). Opioid receptors are coupled to G i /G o proteins to affect several different effectors, including inhibition of adenylyl cyclase, enhancement of K ϩ conductance, decrease in Ca 2ϩ conductance, and activation of p42/p44 mitogen-activated protein kinases (for review, see Law et al., 2000). In addition, opioid receptors are shown to act through Gz to inhibit adenylyl cyclase and G 16 to activate phospholipase C (Lai et al., 1995;Lee et al., 1998), and opioid receptors stimulate Na , ␦, and opioid receptors of several species have been cloned (for review, see Kieffer, 1995;Knapp et al., 1995). In addition, a receptor with high sequence similarity to the opioid receptors, termed the ORL1 receptor, was cloned and found to be coupled to G i /G o proteins (for review, see Kieffer, 1995;Knapp et al., 1995). Subsequently, the endogenous ligand for the ORL1 receptor was identified and named noThis work was supported by National I...
Background Previous studies have demonstrated that isoflurane can provide both neuroprotection and neurotoxicity in various tissue culture models and in rodent developing brains. The cellular and molecular mechanisms mediating these dual effects are not clear, but the exposure level and duration of isoflurane appear to be determinant factors. Methods Using the ReNcell CX human neural progenitor cell line, we investigated the impact of prolonged exposure to varying isoflurane concentrations on cell survival and neurogenesis. In addition, we assessed the impact of short isoflurane preconditioning on elevation of cytosolic Ca2+ concentration and cytotoxic effects mediated by prolonged isoflurane exposures and the contribution of InsP3 or ryanodine receptors activation to these processes. Results Short exposures to low isoflurane concentrations promote proliferation and differentiation of ReNcell CX cells, with no cell damage. However, prolonged exposures to high isoflurane concentrations induced significant ReNcell CX cell damage and inhibited cell proliferation. These prolonged exposures suppressed neuronal cell fate, while promoting glial cell fate. Preconditioning of ReNcell CX cultures with short exposures to low concentrations of isoflurane ameliorated the effects of prolonged exposures to isoflurane. Pretreatment of ReNcell cultures with InsP3 or ryanodine receptor antagonists mostly prevented isoflurane-mediated effects on survival, proliferation, and differentiation. Finally, isoflurane preconditioned cultures showed significantly less isoflurane-evoked changes in calcium concentration. Conclusion The commonly used general anesthetic isoflurane exerts dual effects on neuronal stem cell survival, proliferation and differentiation, which may be attributed to differential regulation of calcium release through activation of endoplasmic reticulum localized InsP3 and/or ryanodine receptors.
Several chemically diverse pruritogens, including bombesin, compound 48/80, norbinaltorphimine, and 5'-GNTI, cause rodents to scratch excessively in a stable, uniform manner and consequently provide convenient animal models of itch against which potential antipruritics may be evaluated, structure-activity relationships established, and the nature of spontaneous, repetitive behavior itself analyzed. Decreasing the number of scratching bouts in these apparently simple models has been the requisite first step in the progress of kappa opioid agonists such as nalbuphine, asimadoline, and CR845 toward clinical testing as antipruritics. Nalfurafine is the prime example of a kappa agonist spanning the developmental divide between scratching mice models and commercialization within 10 years. Patients undergoing hemodialysis and suffering from the itching associated with uremic pruritus, and potentially those inflicted with atopic dermatitis, are the beneficiaries.
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